Method for identifying substances useful for treating inflammation using the response element to the ikbalpha ROR receptor

ABSTRACT

The present invention concerns a method for identifying substances useful for treating inflammation, characterized in that it comprises the following steps: (a) placing said substance in contact with a nucleic acid construct containing the Retinoid-Related Orphan Receptor (ROR) receptor response element of the promoter of the IκBα gene, under conditions suitable for allowing said substance to bind to said response element, (b) measuring the possible binding of said substance to the response element or the transcriptional activity of the response element or of a promoter containing it, (c) optionally comparing the measurement of step (b) with a measurement carried out under conditions similar to those of steps (a) and (b) but with a nucleic acid construct containing the mutated response element.

This application is the US national phase of international applicationPCT/FR01/02652 filed 22 Aug. 2001 which designated the U.S.

The present invention relates to the use of the ROR receptor responseelement of the IκBα gene, and more specifically the sequence −910/−898of the promoter of this gene, for screening substances useful fortreating or preventing inflammation. In a particular embodiment, theinvention concerns the use of a ROR receptor and/or at least oneresponse element thereof or a functional equivalent thereof forscreening substances useful for treating or preventing inflammation.

The NF-κB complex is composed of a wide variety of protein dimers fromthe NF-κB/ReI family. Five proteins from this family have beenidentified in mammals: NF-κB1 (p50 and its precursor p105), NF-κB2 (p52and its precursor p100), c-ReI, ReIA/p65 and ReIB (Ghosh, S. et al.,Ann. Rev. Immunol. 1998, 16: 225-60). All these proteins contain ahighly conserved domain, called RHD for ReI Homology Domain, composed ofabout 300 amino acids with two “Ig-like” domains. This RHD domain isinvolved in the dimerization of NF-κB proteins, binding to DNA, andinteraction with IκBs proteins and it posseses a nuclear localizationsignal (NLS). NF-κB dimers display different affinities for recognizingthe consensus sequence GGGRNNYYCC, wherein R is a purine base, Y apyrimidine base and N any base (Miyamoto, S. Verma, I. M., Adv. CancerRes. 1995, 66: 255-92). Proteins from the NF-κB/ReI family also showdifferent abilities for activating transcription; only the p65 and c-ReIproteins are capable of doing so, although ReIB activates transcriptionin certain cell types. Knocking out genes encoding the differentproteins in this family has revealed different functions specific ofeach of its members. The p50/p65 heterodimer was the first dimer to beidentified and it is the most abundant in the majority of cell types.

The NF-κB pathway regulates the transcription of a large number of genesinvolved in the immune and inflammatory response (Bauerle, P. A. andBaichwal, V. R., Adv. Immunol. 1997, 65: 111-37; Barnes, P. J. andKarin, M., N. Engl. J. Med. 1997, Ap 10, 336 (15): 1066-71; Siebenlist,U. et al., Ann. Rev. Cell Biol. 1994, 10: 405-55). A deregulation ofthis signalling pathway underlies many diseases such as cancer,neurodegenerative disorders, arthritis and asthma.

The NF-κB signalling pathway is activated by a broad spectrum ofstimuli: cytokines (TNFα, IL-1), mitogens, lipopolysaccharide, viruses,viral proteins, double stranded RNA, physical and chemical stresses(Ghosh, S. et al., Ann. Rev. Immunol. 1998, 16: 225-60; Siebenlist, U.et al., Ann. Rev. Cell Biol. 1994, 10: 405-55).

In the majority of inactivated cells, NF-κB is present as an inactivecytoplasmic complex in which it is bound to an inhibitory protein IκB(Forman B. M. and Baltimore, D., Science 1998, 242: 540-546). Activationof NF-κB occurs through activation of an IκB kinase (IKK) whichphosphorylates the IκBα protein at serine 32 and serine 36, leading todegradation of IκBα and release of NF-κB p52 and p65 proteins (Bauerle,P. A. and Baltimore D., Cell 1996, 87: 13-20; Verma, I. M., Stevenson,J. K., Schwartz, E. M., Van Antwerp, D. and Miyamoto, S., Genes & Dev.1995, 9: 2723-2735). The NF-κB dimers thus released migrate to thenucleus where they regulate transcription.

The IκB family comprises the proteins IκBα, IκBβ, IκBγ, IκBε, Bcl-3,NF-κB1, NF-κB2 (Whiteside, S. T. and Israel, A., Semin. Cancer Biol.1997, Apr 8(2): 75-82). IκB proteins are involved in the terminal stageof the NF-κB signalling pathway since these newly synthesized proteinsare directed to the nucleus where they interact with DNA-bound NF-κBcomplexes, causing dissociation from the binding site and translocationback into the cytoplasm (Arenzana-Seeisdedos, F. et al., J. Cell Sci.1997, Feb. 110: 369-78).

Expression of the IκBα protein is controlled by the NF-κB pathway whichexerts feedback control of this signalling pathway (Sun, S. C. et al.,Science 1993, Mar 26, 259 (5103): 1912-5). In addition, glucocorticoidsalso appear to control IκBα expression (Auphan, N. et al., 1995, Oct.13, 270 (5234): 286-90; Scheinman, R. I., 1995, Oct. 13, 270 (5234):283-6), while estrogens reduce expression levels (Sun S. C. et al.,Science 1993, Mar. 26, 259 (5103): 1912-5). Lastly, functional analysisof the IκBα promoter has revealed the critical role of an AP-1 bindingsite (Kralova, J. et al., Oncogene 1996, Jun. 20, 12 (12): 2595-604) andan SP1 binding site (Algarte, M. et al., Mol. Cell Biol., 1999, Sep. 19(9): 64140-53).

The RORα receptor (Retinoid-related orphan receptor alpha) (NR1F1) is amember of the nuclear receptor family which binds as a monomer, via anRORE response element composed of a single 6 base pair sequence with thehemipalindromic motif AGGTCA preceded by an A/T-rich region (Giguère,V., McBroom, L. B. D. and Flock, G., Mol. Cell. Biol., 1995, 15:2517-2526; McBroom, L. B. D., Flock, G. and Giguère, V., Mol. Cell.Biol., 1995, 15: 796-808). Under certain conditions RORα can also bindto DNA in homodimeric form via a response element composed of arepetition of two hemipalindromic motifs AGGTCA separated by twonucleotides and preceded by an A/T-rich region (Harding, H. P. et al.,Mol. Endocrinol. 1997, Oct. 11 (11): 1737-46).

RORE sites are ROR response elements on which the ROR receptor binds tomodulate the transcriptional activity of the gene situated downstream.Such sites may be used to confer ROR sensitivity to a heterologouspromoter.

The RORα gene has four isoforms sharing a common DNA binding domain(DBD) as well as a common ligand binding domain (LBD). These isoformsdiffer at their amino terminus (Giguère, V. et al., Genes & Dev. 1994,8: 538-553). RORα-deficient mice display cerebral defects (ataxia)characteristic of staggerer mice, which harbor a deletion in the RORαgene that creates a truncated protein unable to bind DNA (Hamilton, B.A. et al., Nature 1996, 379: 736-739†; D'Ambrosio, D. et al., J. Clin.Invest. 1998, 101: 252-262; Dussault, I., Fawcett, D., Mathyssen, A.,Bader, J.-A. and Giguère, V., Mech. Dev. 1998, 70: 147-153; Steinmayr,M. et al., J. Biol. Chem. 1998, 95: 3960-3965). Furthermore, staggerermice overproduce IL-1 (Kopmels, B. et al., J. Neurochem. 1992, 58:192-199).

It has been shown in the prior art that staggerer mice bearing amutation in the RORα gene (mutation which prevents translation of theDNA binding domain) have immune defects such as hyperproduction of IL-1αin macrophages (Kopmels, B. et al., J. Neurochem. 1992, 58: 192-199;Kopmels, B. et al., J. Neurochem. 1990, 55: 1980-1985).

In another study, the RORα receptor, which belongs to the orphan nuclearreceptor family, was reported to be activated by CGP 52608, athiazolidinedione derivative, in Drosophila SL3 cells (Wiesenberg, I. etal., Mol. Pharmacol. 1998, 53: 1131-1138). This study also showed thatCGP 52608 exerts anti-arthritic activity in vivo. However, it did notprovide any evidence demonstrating that the anti-inflammatory activityof CGP 52608 is ROR-dependent in mammalian cells.

It has also been suggested, based solely on the presence of an RORresponse element in its promoter, that the gene encoding 5-lipoxygenase,an important enzyme involved in the control of inflammation and allergy,is a target of RORα (Steinhilber et al., J. Biol. Chem. 1995, 270 (13):7037-7040).

The research carried out within the scope of the present invention hasrevealed a new function of the RORα1 receptor. In a surprising manner,the inventors demonstrated for the first time that RORα negativelyregulates the inflammatory response by interfering with the NF-κBpathway. In fact, it has now been shown that transcription of the IKBαprotein, the main inhibitor of the NF-κB pathway, is activated by RORαvia a new RORE response element, identified in the promoter of the IKBαgene. This response element is a perfect RORE consensus site and islocated between positions −910/-898 of the IKBα promoter, the sitehaving the sequence gagcacAATGTAGGTCAgatag (SEQ ID NO:1) (Ito, C.Y.,Kazantsev, A.G. and Baldwin, A.S., Nucl. Acid Res. 1994, 22: 3787-3792).

Moreover, the inventors showed that mutation of this sequence results ina loss of effect of RORα1, illustrating its functional importance. Anyother nuclear receptor or any other substance which binds to thissequence can therefore modulate IκBα expression.

Now, by binding to nuclear factor NF-κB, the IκBα protein forms aninactive complex (IκBα-NF-κB) which prevents the activation ofexpression of pro-inflammatory genes such as the genes encoding certaincytokines or the COX-2 gene. These data demonstrate that RORα1 or anyother nuclear factor that binds to the sequence −910/−898 of the IκBαpromoter is a potential target for treating chronic inflammatorydisorders.

FIG. 1, attached, outlines the mechanisms involved in regulation of theinflammatory response identified in the present invention.

Demonstration of the role of sequence −910/−898 has therefore allowedthe development of new methods for identifying substances useful fortreating human (chronic) inflammatory disorders such as atherosclerosisor rheumatoid arthritis. It is understood that the sequence identifiedin the invention can also be used for identifying new nuclear factorswhich affect the activity of the IκB promoter through this sequence and,consequently, may be useful for treating human (chronic) inflammatorydisorders such as atherosclerosis or rheumatoid arthritis.

Identification of IκBα as a target of RORα1 has therefore equallyallowed the development of new methods for identifying substances usefulfor treating human (chronic) inflammatory disorders such asatherosclerosis or rheumatoid arthritis.

An object of the present invention therefore resides in a method foridentifying substances useful for treating inflammation, wherein saidmethod comprises the following steps:

a) placing said substance in contact with a nucleic acid constructcontaining the ROR receptor response element of the promoter of the IκBαgene, under conditions suitable for allowing said substance to bind saidresponse element,

b) measuring the possible binding of said substance to the responseelement or the transcriptional activity of the response element or of apromoter containing it,

c) optionally comparing the measurement of step (b) with a measurementcarried out under the same conditions as steps (a) and (b) but with anucleic acid construct containing the mutated response element.

The response element is preferably the RORα receptor response elementand preferably the hRORα receptor.

According to a specific embodiment of the invention, the conditionsallowing said substance to bind said response element comprise thepresence of the ROR receptor or a functional equivalent thereof.

Thus, the method of the invention comprises the following steps:

a) placing said substance in contact with the ROR receptor or afunctional equivalent thereof and a nucleic acid construct containingthe response element to said receptor of the IκBα gene promoter, underconditions suitable for allowing said receptor or its functionalequivalent, said substance or complex formed of the receptor or itsfunctional equivalent and said substance, to bind to said responseelement,

b) measuring the possible binding of the ROR receptor or its functionalequivalent, of the substance or a complex formed of the ROR receptor orits functional equivalent and said substance, to the response element orthe transcriptional activity of the response element or of a promotercontaining it,

c) optionally comparing the measurement of step (b) with a measurementcarried out under the same conditions as steps (a) and (b) but with anucleic acid construct containing the mutated response element.

Functional equivalent of the ROR receptor is understood to mean anynuclear factor able to bind to the ROR receptor response element of thepromoter of the IκBα gene. This therefore comprises any proteinsimultaneously containing:

-   -   a binding site having a selectivity similar to that of ROR for a        given ligand of the latter and,    -   a DNA binding site recognizing the same response element as ROR        or a response element having a similar nucleic acid sequence.

The term “functional equivalent” which refers to the ROR receptorgenerally designates any polypeptide derived from the structure of theROR receptor and retaining the ability to bind to the response elementof the IκBα gene, particularly any response element having the sequenceSEQ. ID. NO: 1 or functional variants thereof. The functionalequivalents may be natural variants (polymorphism, splicing, etc.),fragments, mutants, deletants, etc. Preferably, they are polypeptidescomprising at least one amino acid region having at least 60% identity,preferably at least 75% and even more preferably at least 90 or 95%, tothat of the ROR receptor, particularly RORα, preferably hRORα. The termfurther includes polypeptides containing the DNA binding site of the RORreceptor.

Thus, within the context of the present invention, the ROR receptorrefers to all the isoforms α, β and γ of the ROR family. The RORαreceptor is more particularly preferred and the hRORα receptor is evenmore preferred.

A functional equivalent of the ROR receptor may also be a nuclear factoridentified by the method of the invention described hereinabove.

An advantageous embodiment of the method of the invention consists inusing in step (a) a nucleic acid construct which contains several copiesof the RORα response element and preferably the hRORα receptor of theIκBα gene promoter. For instance the contruct may contain 1 to 10 copiesof the RORα receptor response element, typically 1 to 5 copies.

An advantageous embodiment of the method of the invention consists inusing in step (a) a nucleic acid construct comprising all or part of theIκBα gene promoter containing the ROR receptor response element.

A preferred example of a nucleic acid construct according to step (a)comprises or is constituted by the sequence between positions −910/−898of the IκBα promoter which has the following sequence:

GAGCACAATGTAGGTCAGATAG (SEQ ID No: 1)

or a fragment thereof containing but not restricted to the 6 base pairconsensus sequence underlined above. Examples of ROR receptor responseelement constructs that may be used in the invention comprise thefollowing specific sequences:

TAGGTCAG (SEQ ID NO: 6) GTAGGTCAGA (SEQ ID NO: 7) ATGTAGGTCAGATA (SEQ IDNO: 8)

In a particular embodiment, a nucleic acid is used comprising the entiresequence SEQ ID NO: 1. Compared with the 6 base pair consensus sequencealone underlined in SEQ ID No: 1 hereinabove, this sequence has theadvantage of including one or more regions involved in interactions withnuclear factors.

The possible binding of the substance, the ROR receptor or a complexformed of the ROR receptor and said substance to the response elementmay be measured by any method known to those skilled in the art, forinstance by detecting a signal produced by the response elementfollowing said binding. These may be any direct or indirect methods,such as those which use a reporter gene, binding tests, etc.

Thus, a preferred embodiment of the invention consists in using anucleic acid construct combining the response element of the IκBα genewith a reporter gene. In an advantageous manner, said reporter gene isplaced under the control of a promoter containing at least one copy ofsaid response element.

The reporter gene may therefore be placed under the control of anypromoter whose sequence comprises the sequence SEQ. ID. NO: 1 or afunctional variant thereof. Such specific sequence may be present in oneor more copies in the promoter (preferably 1 to 10 and even morepreferably 1 to 6), upstream, downstream or internally, in the sameorientation or in the opposite orientation. In a preferred embodiment ofthe invention, the reporter gene is placed under the control of apromoter comprising one or more copies of the sequence SEQ ID NO: 1 or afragment thereof containing at least the consensus region. It ispreferably a promoter having differential activity in the absence andpresence of ROR or a functional equivalent which can be detected.

To create a promoter according to the invention, the ROR receptorresponse element may be associated with a transcriptional minimalpromoter. The minimal promoter is a transcriptional promoter having alow or nonexistent basal activity, and which can be increased in thepresence of a transcriptional activator (e.g. ROR). A minimal promotermay therefore be a naturally weak promoter in mammalian cells, i.e.producing a non-toxic and/or insufficient expression to cause a markedbiological effect. In an advantageous manner, a minimal promoter is aconstruct prepared from a native promoter, by deleting region(s) thatare not essential for transcriptional activity. For instance, it ispreferably a promoter comprising principally a TATA box, generally lessthan 160 nucleotides in size, centered around the transcriptioninitiation codon. A minimal promoter may thus be prepared from strong orweak viral or cellular promoters, such as for example the promoter ofthe herpes virus thymidine kinase (TK) gene, the CMV early promoter, thePGK promoter, the SV40 promoter, etc.

In a preferred embodiment, the reporter gene is placed under the controlof the promoter of the IκBα gene.

Any reporter gene allowing to measure the activity of nuclear receptorson the sequence comprising their response element may be used in thescreening method according to the invention. These are exemplified forinstance by the chloramphenicol acetyltransferase (CAT) gene, thefirefly (Luc) or Renilla (Ren) luciferase gene, the secreted alkalinephosphatase gene (SAP) or that of beta-galactosidase (β-Gal) and theIκBα gene itself.

The activity of the proteins encoded by these genes can be easilymeasured by conventional methods and gives an indirect idea of theeffect of the nuclear receptors on gene expression by measuring theamount of proteins produced and/or their enzymatic activity.

According to a more particular embodiment of the invention, the reportergene is the IκBα gene. In this case, the accumulation of IκBα proteincan be measured by its ability to bind NF-κB or to inhibit its actionfor example by using a reporter vector comprising several NF-κB responseelements cloned upstream of a reporter gene.

The use of a reporter gene has the advantage of verifying whether thereceptor or a functional equivalent thereof or said substance is ablenot only to bind, alone or complexed with the ROR receptor, to saidresponse element, but also to increase the transcriptional activity ofthe response element or of a promoter containing said response element.In this manner the role of said substance in inhibiting the inflammatoryresponse can be confirmed.

Advantageously, this ability to increase transcriptional activity may bemeasured at step (b) and possibly (c) on the IκB gene by any direct orindirect methods known to those skilled in the art, such astransfection, analysis of mRNA or protein in vitro and in in vitro andin vivo models.

An example of the method according to the invention comprises thefollowing steps:

a) placing said substance in contact with a host cell transformed with anucleic acid construct comprising at least a reporter gene functionallycombined with the ROR receptor response element of the IκBα genepromoter as defined hereinabove,

b) measuring the expression of the reporter gene by any suitable means,

c) optionally comparing the measurement of step (b) with a measurementcarried out under the same conditions as steps (a) and (b) but with anucleic acid construct containing the mutated response element.

According to a specific embodiment of the hereinabove method, the hostcell of step (a) expresses an ROR receptor or a functional equivalentthereof as defined hereinabove.

In step (c), comparison with the measurement of step (b) isadvantageously carried out with a measurement under the same conditionsas steps (a) and (b) but with a nucleic acid construct containing themutated response element, i.e. an ROR response element whose sequencehas been altered so as to make it unable to bind the ROR receptor in afunctional manner. Such mutated response element advantageously has thefollowing sequence GAGCACAATGTXXXXXXGATAG (SEQ ID NO:16), wherein X ischosen from among A, T, C, G.

As a non-limiting example, the following mutated sequences arepreferred:

GAGCACAATGTATTTCAGATAG (SEQ ID NO: 9) GAGCACAATGTAAATCAGATAG (SEQ ID NO:10) GAGCACAATGTACATCAGATAG (SEQ ID NO: 11) GAGCACAATGTAACTCAGATAG (SEQID NO: 12) GAGCACAATGTATATCAGATAG (SEQ ID NO: 13) GAGCACAATGTAATTCAGATAG(SEQ ID NO: 14) GAGCACAATGTACCTCAGATAG (SEQ ID NO: 15)

The substances that can be identified by the method according to theinvention may be chemical or biological compounds. For instance, theymay be nuclear factors.

Furthermore, classical methods for identifying clones encodingDNA-binding proteins may be used in the method of the invention. Forexample, it is possible to use the screening of a λgt11 expressionlibrary (Ausubel et al., Current Protocols in Molecular Biology, JohnWiley & Sons, Inc. 1997), purification by affinity chromatographyfollowed by sequencing the protein (Ausubel et al., Current Protocols inMolecular Biology, John Wiley & Sons, Inc. 1997), the so-called“one-hybrid” method (Wang, M. M., Nature 1993, Jul. 8, 364 (6433):121-6), or yet the so-called “phage display” method (Smith, G. P. andScott, J. K., Methods Enzymol. 1993, 217†: 228-57; O'Neil, K. T. andHoess, R. T., Curr. Opin Struct. Biol. 1995, Aug. 5(4)†: 443-9).

The invention equally concerns a vector comprising at least one nucleicacid construct containing at least one copy of the response element ofthe IκBα gene mutated or not, able to be used in a method foridentifying substances described hereinabove.

The invention further relates to the host cells containing such avector.

Host cell is understood to mean any type of cell suitable for expressingthe abovedescribed genes, such as in particular mammalian cells,bacteria or yeasts or even insect cells. In a preferred embodiment ofthe invention, the host cells are mammalian cells (hepatocytes,fibroblasts, endothelial cells, muscle cells, etc.). Even morepreferably, such cells may be human cells. They may also be primarycultures or established cell lines. In another embodiment, it is furtherpossible to use prokaryotic cells (bacteria), yeast cells(Saccharomyces, Kluyveromyces, etc.), plant cells, etc.

The substances may be placed in contact with the cells at differenttimes, according to their effect(s), their concentration, the cell typeand the technical factors.

The vectors used are, of course, suited to the type of cell beingtransfected, and include plasmids, viruses or artificial chromosomes.

The test substance may be placed in contact with the nucleic acidconstruct or cells on (or in) any suitable support and notably on aplate, in a tube or flask, a membrane, etc. In general, placing incontact occurs in a multiwell plate which makes it possible to conductmany different tests in parallel. Typical supports includemicrotitration plates and more particularly plates having 96 or 384wells (or more) which are easy to manipulate.

Depending on the support and the nature of the test substance, differentquantities of cells may be used to implement the described methods.Typically, 10³ to 10⁶ are placed in contact with a type of testsubstance, in a suitable culture medium, and preferably between 10⁴ and10⁵ cells.

The quantity (or concentration) of test substance may be adjusted by theuser according to the type of substance (its toxicity, its ability topenetrate the cells, etc.), the number of cells, the incubation time,etc. Generally, the cells are exposed to quantities of test substanceranging from 1 nM to 1 mM. Of course other concentrations may be testedwithout deviating from the present invention. Moreover, each substancemay be tested in parallel at different concentrations. Also, differentadjuvants and/or vectors and/or products that facilitate penetration ofthe substances inside the cells such as liposomes, cationic lipids orpolymers may, furthermore, be used, where necessary. Contact time mayrange for example from a few minutes to a few hours or days,particularly between 5 and 72 hours, generally between 12 and 48 hours.The cells and the various reagents must preferably remain in contactlong enough to allow de novo synthesis of the product expressed by thereporter gene or the interaction under study.

The invention equally concerns substances identified through the methodof the invention with the exception of the ROR receptor itself.

The invention therefore also has as its object a pharmaceuticalcomposition for preventing or treating inflammatory disorders,comprising as active principle at least one substance other than the RORreceptor that can bind, alone or complexed with the ROR receptor or afunctional equivalent thereof, to the ROR receptor response element ofthe IκBα gene.

The invention more particularly concerns the use of a transcriptionalactivator of the IκBα gene for preparing a medicinal product intended totreat or prevent inflammatory disorders.

According to a particular manner of use according to the invention, thetranscriptional activator of the IκBα gene is a substance able to bindalone or complexed with the ROR receptor to the response element to saidROR receptor of the IκBα gene. The transcriptional activator of the IκBαgene may therefore be an activator of the binding of RORα to theresponse element of the IκBα gene promoter or of its transcriptionalactivity.

The invention therefore relates more particularly to the use of asubstance able to bind, alone or complexed with the ROR receptor or afunctional equivalent thereof, to the IκBα gene promoter responseelement and to increase the expression of IκBα for preparing a medicinalproduct intended to treat or prevent inflammatory disorders.

Other advantages and features of the invention will become apparent inthe following examples which refer to the attached figures wherein:

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 outlines the mechanisms involved in the regulation of theinflammatory response identified in the present invention

FIGS. 2A and 2B depict RORα expression in different types of cells inthe vascular wall based on RT-PCR analysis (35 cycles) of RORα (FIG. 2A)and GAPDH (FIG. 2B) mRNA (C-PCR and C-RT are negative controls for thePCR and RT, respectively).

FIG. 2C shows the RT-PCR analysis of RORα mRNA in smooth muscle cellsafter a 24 hour infection with Ad-RORα1 or Ad-GFP.

FIG. 2D illustrates the analysis of RORα protein expression in smoothmuscle cells infected for 24 hours with or without Ad-RORα1.

The immunocytochemical studies were performed as described previously(Chinetti, G. et al., J. Biol. Chem. 1980, 273: 25573-25580) using arabbit polyclonal RORα antibody directed against amino acids 163 to 225.

FIGS. 3A and 3B show that RORα1 inhibits the secretion of IL-6 (FIG.3A), IL-8 (FIG. 3B) and COX-2 expression induced by TNFα in smoothmuscle cells. Primary smooth muscle cells were infected with Ad-RORα1 orAd-GFP virus for 16 hours, then subjected to 24 hour stimulation withTNFα (10 ng/ml). After this treatment, IL-6 and IL-8 concentrations inthe medium were determined by ELISA (R&D Systems UK).

FIGS. 3C and 3D illustrate the expression of COX2 and RORα proteindetermined by Western blot analysis. COX2 protein levels were normalizedto β-actin content in the cells (NS=nonspecific).

FIGS. 4A to 4C depict the inhibition of NF-κB activation by RORα1.

FIG. 4A shows PAC1A cells transfected with the reporter gene (NF-κB)3-Luc (100 ng) in the presence of an RORα1 expression vector(pSG5-RORα1, 50 ng) or an empty vector (pSG5 control). Two hourspost-transfection, cells were placed in DMEM medium supplemented with0.2% FCS in the presence or absence of LPS (10 mg/ml) for 24 hours.

FIG. 4B shows primary smooth muscle cells infected with Ad-RORα1 orAd-GFP for 16 hours, then stimulated with TNFα (10 ng/ml) for 0, 15, 30and 60 minutes. Total (T; lower gel) and nuclear (N; upper gel) proteinextracts were prepared and analyzed by Western blot using anti-p65antibody (Santa Cruz).

FIG. 4C shows primary smooth muscle cells infected with Ad-RORα1 orAd-GFP for 16 hours, then stimulated with TNFα (10 ng /ml) for 30minutes. Nuclear protein extracts (5 mg) were prepared and analyzed byEMSA using NF-κB and Sp1 labelled probes (Promega). Complexes whichmigrated are indicated by an arrow. The double arrow shows the unboundprobe.

FIGS. 5A and 5B illustrate the induction of IκBα transcription by theRORα1 receptor in cells infected with an RORα expression vector(pSG5-RORα1).

FIG. 5A shows primary smooth muscle cells infected with Ad-RORα1 orAd-GFP for 16 hours. Total RNA was prepared and IκBα and GAPDH mRNAswere measured by Northern blot.

FIG. 5B depicts PAC1A cells transfected with fragments of the IκBαpromoter (100 ng) in the presence of pSG5-RORα1 or empty vector (pSG5,50 ng).

FIGS. 6A to 6C show the specific transcriptional activation of the IκBαgene by RORα1.

RORα1, but not RORα2 or RORα3, binds to an RORE site within the IκBαpromoter.

FIG. 6A shows PAC1A cells transfected with the reporter vector(IκBα-RORE) 2-TK-Luc (100 ng) in the presence of RORα1, RORα2 or RORα3(50 ng) or empty vector pSG5 (50 ng).

FIG. 6B shows PAC1A cells transfected with the reporter vector(IκBα-RORE) 2-TK-Luc (100 ng) in the presence of RORα1 (50 ng),RORα1Δ235 or empty vector (50 ng).

FIG. 6C depicts the gel shift experiments conducted by using the invitro translated RORαl protein (TNT-T7 Promega) and an IκBα-RORElabelled probe. Increasing concentrations (1×, 10× and 100×) of thenative (AGGTCA) or mutated (ACCTCA) oligonucleotide competitor were usedto demonstrate the specificity of the migrating complex. To enhance themobility of the complex, 1 μl of RORα1 antibody was added to the bindingreaction (S: shift of the complex; SS: super-shift of the complex; FP:unbound probe).

I—Materials and Methods

1) Cell Culture

Human aorta smooth muscle cells (PromoCell, Heidelberg, Germany) andhuman coronary artery endothelial cells (Clonetics) were maintained inprimary culture in the usual conditions. Cells were used betweenpassages 5 to 8. Human macrophages and monocytes were prepared andmaintained in primary culture as described previously.

2) RNA Analysis

RNAs were prepared and analyzed by Northern blot as described previouslyusing fragments of IκBα and GAPDH cDNA as probes. For the RT-PCRanalysis of RORα expression, total RNA was transcribed by reversetranscriptase and then amplified by PCR using the following primers:

-   -   for RORα, 5′-GTCAGCAGCTTCTACCTGGAC-3′ (SEQ ID No: 2) and        5′-GTGTTGTTCTGAGAGTGAAAGGCACG-3′ (SEQ ID No: 3) (fragment size:        482 bp);    -   for GAPDH, 5′-ATGCAGCCCCGAATGCTCCTCATCGTGGCC-3′ (SEQ ID No: 4)        and 5′-TTCTTGGAGGCCATGTGGGCCAT-3′ (SEQ ID No: 5) (fragment size:        239 bp)

3) Adenovirus Production

Recombinant adenoviruses Ad-GFP and Ad-RORα1 were obtained by homologousrecombination in E. coli after inserting the cDNAs in the vectorpADCMV2. Stocks of viral particles were obtained as previouslydescribed. The titer of the suspension was determined by counting lyticplaques after infection of 293 cells and defined as pfu/ml. Cells wereinfected at a multiplicity of infection of 100 viral particles per cellby adding the viral stocks directly to the smooth muscle cell culturemedium.

4) Plasmids and Transient Transfection

The expression vectors pSG5-hRORα1, -hRORα2, -hRORα3 and -RORαΔ235 werederived from pCMX vectors described previously. A fragment correspondingto the region −929 /+22 of the IκBα promoter was amplified from humangenomic DNA and cloned into Promega basic pGL2 plasmid to give theconstruct p(−922/+22) IκBα Luc. The mutation of the ROR site present inthis fragment (AGGTCA mutated to ACCTCA) was introduced by site directedmutagenesis (Stratagene). The reporter vector p(−385/+22) IκBα was akind gift of Dr. Israel (Pasteur Institute, Paris). The reporter vector(IκBα-RORE) 2-Tk-Luc was created by inserting two copies of the ROREsite of the IκBα promoter upstream of the minimal promoter of the herpessimplex virus thymidine kinase gene. The construct (NF-κB) 3-Luc wasobtained from Stratagene. PAC1A cells (rat aorta smooth muscle cellline) were transfected using a cationic lipid as previously described.

II—Results

Considering that chronic inflammation is a feature of atherosclerosis(Ross, R., New Engl. J. Med. 1999, 340: 115-126) and that staggerer micehave a pro-inflammatory profile, the inventors measured ROR expressionin the different types of cells in the vascular wall by RT-PCR.

RORα expression was detected in endothelial cells, smooth muscle cells(SMC) and monocytes (FIG. 2A). On the other hand, RORα expression wasfound to be much lower in monocyte-derived macrophages.

To examine the potential role of RORα in regulating the inflammatoryresponse, the inventors generated by homologous recombination anadenovirus vector encoding the RORα1 isoform, which is preponderant(Forman, B. M et al., Mol. Endocrin. 1994, 8: 1253-1261). Infection ofprimary SMC with Ad-RORα1 induced the expression of RORα, whereasinfection with Ad-GFP used as negative control had no effect onendogenous levels (FIG. 2C). The immunocytochemical studies with apolyclonal anti-RORα antibody (aminoacids 163-225) confirmed theseresults at the protein level and furthermore demonstrated that bothendogenous RORα and exogenous RORα are localized primarily in thenucleus (FIG. 2D).

The effects of RORα1 on the different markers of the vascularinflammatory response induced by TNFα were then investigated.

In the absence of stimulation, both IL-6 and IL-8 were detected in cellsinfected with Ad-GFP, an effect due to the immunogenicity of thisconstruct. This weak induction was significantly inhibited by infectionwith Ad-RORα1 (FIGS. 3A and 3B). Treatment of Ad-GFP-infected cells withTNFα (24 h) led to a marked induction of both cytokines which could besignificantly inhibited by infection with Ad-RORα1 (FIGS. 3A and 3B).Endogenous RORα protein was not detected in Ad-GFP-infected cells, whichis undoubtedly due to the weak affinity of the antibody used. However,in cells infected with Ad-RORα1, RORα expression was increased, and thelevel was unaffected by TNFα stimulation (FIG. 3C). COX-2 protein levelswere also measured in the same experiments. TNFα strongly induced COX-2expression, an effect which was abolished in Ad-RORα1-infected cells(FIG. 3D). These data demonstrate that RORα1 negatively regulates theinflammatory response induced by TNFα in primary aorta smooth musclecells.

Within the scope of the research which led to the invention, PAC1A cells(rat smooth muscle cell line) were transiently transfected with apromoter under the control of the NF-κB pathway, in the presence orabsence of RORα1. Considering that these cells do not respond to TNFαstimulation, the inventors used LPS as inducer of the NF-κB pathway. LPStreatment significantly increased (more than 3-fold) the activity of thepromoter under NF-κB control. This induction was reduced in cellscotransfected with RORα1. Basal promoter activity was not significantlyaffected in these conditions (FIG. 4A). These results confirm that RORα1exerts its anti-inflammatory effect by inhibiting the transcriptionalactivity of NF-κB.

The inventors then investigated the effect of RORα1 overexpression onnuclear translocation of p65 by Western blot. They found that treatmentwith TNFα led to transient nuclear translocation of p65 inAd-GFP-infected cells. This translocation was less pronounced in cellsinfected with Ad-RORα1, indicating that RORα1 controls p65 translocationwithout regulating the total expression of p65 protein (FIG. 4B). Gelshift experiments (EMSA) were conducted to examine the effects of thisreduction of migration. Twenty-four hours post-infection, TNFα was addedto the cells for 30 minutes. At the end of this treatment period,nuclear extracts were prepared and used to check for the presence ofNF-κB proteins p50 and p65 by gel shift experiments using labelledprobes whose sequences correspond to the NF-κB consensus site.Ad-GFP-infected cells displayed significantly lower binding activity tothe NF-κB site than cells infected with Ad-RORα1, which is consistentwith the previous results (FIGS. 3A and 3B). The inventors observed thatTNFα stimulation led to a large increase in NF-κB binding to itsresponse element, a binding which was strongly reduced inAd-RORα1-infected cells. Neither TNFα, nor RORα1 overexpression had anyeffect on the binding of nuclear factors to an Sp1 probe (FIG. 4C),which indicates that the action of RORα1 is specific to the NF-κBpathway.

It is also known that many nuclear receptors inhibit transcriptioncontrolled by NF-κB and binding to the NF-κB site, by interacting withthe p65 subunit (Göttlicher, M. Heck, S. and Herrlich, P., J. Mol. Med.1998, 76: 480-489). For example, it was shown that cotransfection withp65 led to strong induction of an NF-κB-controlled promoter in PAC1Acells (data not shown). This induction was not affected bycotransfection with RORα1, indicating that, contrary to other nuclearreceptors, RORα1 exerts a negative control of the NF-κB pathway withoutassociating with p65.

In quiescent cells, the IκBα protein sequesters the p50/p65 heterodimerin the form of an inactive cytoplasmic complex. Considering that RORαinhibits p65 translocation and its subsequent binding to the NF-κB site,IκBα expression levels were determined in cells infected with Ad-GFP orwith Ad-RORα1. Ad-GFP-infected cells expressed low levels of IκBα mRNA(FIG. 5A). IκBα transcripts were considerably increased in cellsinfected with Ad-RORα1 (FIG. 5A). These findings indicate that RORα1inhibits the NF-κB pathway by inducing the expression of the IκBα gene.

The fragment of the IκBα gene promoter between positions −929 and +22was amplified by PCR and inserted upstream of the reporter geneluciferase (Ito, C. Y., Kazantsev, A. G. and Baldwin, A. S., Nucl. AcidRes. 1994, 22: 3787-3792). This construct forms the basis of the presentinvention. Cotransfection of this vector and an RORα1 expression vectorinduced an increase in luciferase activity under the control of the−929/+22 promoter but not the −385/+22 promoter. This indicates that theregion regulated by RORα1 is located between positions −929 and −385(FIG. 5B). Sequence analysis of this region revealed the presence of aresponse element (RORE) therein. Mutation of this RORE response elementblocked the effect of RORα (FIG. 5B). This experiment unequivocallydemonstrates the role of this site in RORα1-mediated induction of IκBαtranscription.

Considering that the RORα gene has several isoforms (Giguère, V. et al.,Genes & Dev. 1994, 8: 538-553), the inventors evaluated the effects ofthe isoforms RORα1, RORα2 and RORα3 on constructs under the control oftwo copies of the RORα response element present in the IκBα-RORE genepromoter cloned upstream of a heterologous promoter (TK promoter).

RORα1 strongly induced the activity of the reporter gene, whichdemonstrates that IκBα-RORE can function independently of the promoter.In contrast, RORα2 and RORα3 did not induce the transcription of theconstruct cited above (FIG. 6A), indicating that transcriptionalactivation of IκBα is specific of the RORα1 isoform.

It has been shown that the negative dominant RORαΔ235 missing the LBDdomain inhibits RORα1-induced transcription by competing with the sameDNA binding domain (Mc Broom, L. D. B., Flock, G. and Giguère, V., Mol.Cell. Biol. 1995, 15: 796-808). The effect of this mutant on thepromoter under the control of the IκBα gene RORE inducible by RORα1 wastested. Cotransfection with RORαΔ235 abolished the effect of RORα1 (FIG.6B). This shows that binding of RORα to the promoter is essential forinduction of transcription. Finally, binding of RORα1 to its responseelement RORE was checked by gel shift assays, using in vitro translatedRORα1 protein. Competition experiments with unlabelled probes, native ormutated, or supershift experiments showed unequivocally that RORα1 bindswith high affinity to the RORE site present in the IκBα gene promoter.

Thus, during the research conducted within the scope of the presentinvention, the inventors showed for the first time that overexpressionof RORα1 in aorta smooth muscle cells inhibits the expression of COX-2,IL-6 and IL-8 induced by TNFα. All of these genes are major inflammatorymarkers in atherosclerotic lesions (Ross, R., New Engl. J. Med. 1999,340: 115-126). The results that emerged from this research show thatRORα exerts a protective role against atherosclerosis through a directvascular effect in addition to its role as a modulator of lipoproteinmetabolism (which occurs through its action on the expression of theapolipoprotein A1 gene (Mamontova, A. et al., Circulation 1998, 98:2738-2743). By negatively regulating IL-8, a key chemokine closelyinvolved in the recruitment of monocytes during the early stages ofatherosclerosis (Ross, R., New Engl. J. Med. 1999, 340: 115-126), RORα1can reduce the initiation and progression of atherosclerotic lesions.

1. A vector comprising at least one copy of the Retinoid-related OrphanReceptor (ROR) response element site of the IKBα gene, operably linkedto a minimal promoter wherein the minimal promoter is obtained from apromoter selected from the group consisting of the promoter of theherpes virus thymidine kinase (TK) gene, the cytomegalovirus (CMV) earlypromoter, the phosphoglycerate (PGK) promoter and the SV40 promoter,wherein said ROR response element site consists of SEQ ID NO: 1 or SEQID NO: 16, wherein SEQ ID NO: 16 is a mutated response element relativeto the wild-type sequence SEQ ID NO:
 1. 2. An isolated host cellcomprising the vector according to claim
 1. 3. A method of screeningtest substances to identify candidate substances useful for decreasinginflammation, wherein said method comprises the following steps: a)placing each test substance in contact with a nucleic acid constructcontaining at least one ROR response element site of the IKBα genepromoter, under conditions allowing each test substance to bind to saidat least one response element site, b) identifying the test substanceswhich effectively bind to the response element, c) selecting the testsubstances that bind to the response element as candidate substancesuseful for decreasing inflammation; wherein said ROR response elementsite consists of SEQ ID NO:
 1. 4. A method of screening test substancesto identify candidate substances useful for decreasing inflammation,wherein said method comprises the following steps: a) placing each testsubstance in contact with a nucleic acid construct containing at leastone ROR response element site of the IKBα gene promoter or a promotercontaining said at least one ROR response element site wherein thenucleic acid construct is operably linked to a detectable nucleic acidunder conditions allowing each test substance to bind to said at leastone response element site, b) measuring expression of the operablylinked nucleic acid to identify the test substances which effectivelyincrease the transcriptional activity from the response element site orfrom the promoter; c) selecting the test substances that increase thetranscriptional activity from the response element site or from thepromoter as candidate substances useful for decreasing inflammation;wherein said ROR response element site consists of SEQ ID NO:
 1. 5. Themethod according to claim 3 or 4, wherein in step a) the test substanceis further placed into contact with a ROR and wherein step b) consistsin measuring the possible binding of each test substance to the responseelement.
 6. The method according to claim 5, wherein the measurement ofstep (b) is compared with a measurement carried out under conditions ofstep (a) and (b) but with a nucleic acid having the following sequence:GAGCACAATGTXXXXXXGATAG (SEQ ID NO:16), wherein X is selected from thegroup consisting of A, T, C and G, wherein SEQ ID NO:16 is a mutatedresponse element relative to the wild-type sequence SEQ ID NO:1.
 7. Themethod according to claims 3 or 4, wherein the measurement of step (b)is compared with a measurement carried out under conditions of step (a)and (b) but with a nucleic acid construct having the following sequence:GAGCACAATGTXXXXXXGATAG (SEQ ID NO:16), wherein X is selected from thegroup consisting of A, T, C and G, wherein SEQ ID NO:16 is a mutatedresponse element relative to the wild-type sequence SEQ ID NO:1.
 8. Themethod according to claim 4, wherein at step (a), the operably linkeddetectable nucleic acid is a reporter gene placed under the control ofthe promoter comprising said at least one ROR response element site ofthe IKBα gene promoter.
 9. The method according to claim 8, wherein thereporter gene is selected from the group consisting of an IkBα gene, achloramphenicol acetyltransferase gene, a firefly luciferase gene, aRenilla luciferase gene, a secreted alkaline phosphatase gene, and abeta-galactosidase gene.
 10. The method according to claim 4, wherein atstep (a), the operably linked nucleic acid is a reporter gene placedunder the control the promoter comprising said at least one ROR responseelement site of the IKBα gene promoter, wherein said promoter comprisingsaid at least one ROR response element site of the IKBα gene promoter isthe promoter of an IKBα gene.
 11. The method according to claim 10,wherein the reporter gene is selected from the group consisting of aIKBα gene, a chloramphenicol acetyltransferase gene, a fireflyluciferase gene, a Renilla luciferase gene, a secreted alkalinephosphatase gene and a beta-galactosidase gene.
 12. A method ofscreening test substances to identify candidate substances useful fortreating inflammation, wherein said method comprises the followingsteps: a) placing each test substance in contact with a host celltransformed with a nucleic acid construct containing at least onereporter gene operably linked to a ROR response element site of the IKBαgene promoter or from a promoter containing said at least one RORresponse element site, b) measuring the expression of the reporter geneby any suitable means and so identifying the test substances whicheffectively increase the transcriptional activity from the responseelement site or from the promoter containing said response element siteand potentially bind to the response element, c) selecting the testsubstances that increase the transcriptional activity from the responseelement site or from the promoter containing said response element siteas candidate substances useful for decreasing inflammation; wherein saidRetinoid-related Orphan Receptor (ROR) response element site consists ofSEQ ID NO:1.
 13. The method according to claim 12, wherein the host cellof step (a) expresses a ROR.
 14. The method according to claim 12,wherein the measurement of step (b) is compared with a measurementcarried out under the same conditions as those of steps (a) and (b) butwith a nucleic acid construct having the following sequence:GAGCACAATGTXXXXXXGATAG (SEQ ID NO:16), wherein X is selected from thegroup consisting of A, T, C, and G, wherein SEQ ID NO:16 is a mutatedresponse element relative to the wild-type sequence SEQ ID NO:1.